BIOSCAN QC Report

Statistics for the positive controls

Total number of positive controls: 96 
Number of positive controls per plate: 1 

All plates have positive controls: TRUE  
Total number of reads in positive controls: 27813 
Maximum number of reads: 469 in positive control sample: CONTROL_POS_CAMP_013_G12 
Minimum number of reads: 90 in CONTROL_POS_YARN_031_G12 

Average number of positive control reads: 289.71875 
Median number of positive control reads: 296.5 
Read standard deviation: 71.4152723051663 

Quantiles:
 5%: 134.75
10%: 213
25%: 249.5
50%: 296.5
75%: 329.5
95%: 392.25
100%: 469

Blue solid line: read mean
Orange dotted lines: 5% and 10% lower quantiles

Number of positive control samples in the lower 5% quantile: 5 

 CONTROL_POS_FACE_209_G12
CONTROL_POS_FACE_232_G12
CONTROL_POS_PBRI_001_G12
CONTROL_POS_PBRI_004_G12
CONTROL_POS_YARN_031_G12 

Names of the associated partners: BIFOR, PBRI, YARN

Statistics for the negative controls

Total number of negative controls: 500 
Total number of lysate negative controls: 417 
Total number of empty negative controls: 83 

Number of negative controls per plate:

Number of negative controls per plate	Number of plates
1	7
2	76
more than 2	13


All plates have negative controls: TRUE 

Total number of reads in lysate negative controls: 2096 
Total number of reads in empty negative controls: 62 
Maximum number of reads: 230 in lysate negative control sample: CONTROL_NEG_LYSATE_CAMP_017_H12 
Maximum number of reads: 6 in empty negative control sample: CONTROL_NEG_WWTS_005_A8 

Zero reads in: 286 negative control samples 
In lysate controls: 240 
In empty controls: 46 

Average number of negative control reads: 4.316 
In lysate controls: 5.02637889688249 
In empty controls: 0.746987951807229 

Median number of negative control reads: 0 
In lysate controls: 0 
In empty controls: 0 

Skewness number of negative control reads: 9.3101349995259 
In lysate controls: 8.52792548498113 
In empty controls: 2.17703718332309 

Quantiles in lysate controls:
 5%: 0
10%: 0
25%: 0
50%: 0
75%: 3
95%: 30
98%: 37.68 

Quantiles in empty controls:
 5%: 0
10%: 0
25%: 0
50%: 0
75%: 1
95%: 2
98%: 3.72

Blue solid line: read mean
Orange dotted lines: upper 5% and 2% of samples with the highers number of reads

Number of negative control samples in the higher 5%: 26 
 Out of in the lysate controls: 26 
 Out of in the empty controls: 0 
 
Number of negative control samples in the higher 2%: 10 
 Out of in the lysate controls: 10 
 Out of in the empty controls: 0 

Names of the associated partners: CAMP, BIFOR, NENM, PBRI, WWTS

Statistics for the samples

Number of samples in the batch (exclusing controls): 8620 
Total number of partner plates: 96 
Total number of sample reads: 2813154 

Maximum number of sample reads: 803 in sample: FACE_220_D9 
Minimum number of sample reads: 0 in 336 samples
 which is 3.89791183294664 % of all samples 

Average number of reads: 326.351972157773 
Median number of reads: 362 
Read standard deviation: 152.439735911066 
Skewness number of sample reads: -0.660035204264651 

Quantiles:
 5%: 1
10%: 68.9000000000001
25%: 240
50%: 362
75%: 435
95%: 523
100%: 803

Blue solid line: read mean
Orange dotted lines: lower 5% and 10% of samples

Number of samples in the lower 10%: 862 out of 8620 samples 
Number of samples in the lower 5%: 470 out of 8620 samples 

Partners associated with the bottom 5% of samples by read count:

Partner names	Frequency
BIFOR	170
NENM	102
CAMP	61
YARN	51
WWTS	42
PBRI	40
SNST	4


Number of samples with 0 reads: 336

Plate boxplots

Plates where the 75th percentile of the data is lower than expected mean read count (dark grey):

 CAMP_039
FACE_209
FACE_227
PBRI_005
SNST_005
SNST_007
YARN_026
YARN_031 
 
which constitutes 8.33333333333333 % of all partner plates in this batch

Grey line: median
Brown line: mean
Green data points: positive controls
Blue data points: empty negative controls
Navy data points: lysate negative controls

Plates where the 75th percentile of the data is lower than expected mean read count (dark grey):  
How many samples from the low-performance partner plates are present in the low-performance UMI plates (purple data points): 0 %

Assess the positive controls with the low number of reads detected in the previous steps:

FACE_209 More reads in positive control than in samples on average.
 Observed number of reads: 113 Expected: 105.440860215054 

FACE_232 Positive control failed.
 Observed number of reads: 133 Expected: 225.161290322581 

PBRI_001 Positive control failed.
 Observed number of reads: 131 Expected: 308.591397849462 

PBRI_004 Positive control failed.
 Observed number of reads: 134 Expected: 247.430107526882 

YARN_031 Positive control failed.
 Observed number of reads: 90 Expected: 214.537634408602

YARN_031 
FACE_209

The above plates have lower than expected number of reads 
AND failed positive controls. 
THESE PLATES NEED TO BE EXAMINED FURTHER

Low-quality plates are displayed here. All the other plates are plotted in the last part of this report.
Green squares: controls [any kind]

### Assessment of sequence conflicts and contaminants

Positive control as contamination source

NOTE: All sample and sequence IDs match - data successfully merged

Positive control OTU is TAX:1287025 

Non-positve control samples that contain positive control reads:

Sample	Control Sequence Count	Sequence Similarity	Sequence Type	UMI Plate ID
FACE_240_B3	1	99.23195	secondary	8

Number of samples with positive control OTU as primary sequence: NA 
Number of samples with positive control OTU as secondary sequence: 1 
out of 4842 samples with secondary sequences 

Location of the contaminants relative to the source:

Orange square: positive contros
Green squares: samples with positive control contamination

Read count mean of all secondary sequences in all samples: 5.10170152601043 
Read count mean of all positive control sequences in other samples: 1 

Read count median of all secondary sequences in all samples: 1 
Read count median of all positive control sequences in other samples: 1

Blue solid line: secondary hit read mean
Orange dotted lines: mean of reads found as secondary contaminants from the positive controls in other samples
Both lines should be in close proximity meaning that the secondary contamination from positive controls is comparable to the potential contamination in other samples.

There are no samples that contain positive control reads as the primary hit

NO SAMPLES TO BE REMOVED

NOTE: the above samples are automatically removed if:

There’s only one primary read

Secondary sequence found in the same sample is not an Arthropod

Negative control contamination

Distribution of reads in negative controls

NOTE: contamination source can be either primary or secondary sequence within samples!

Family	No. Source Samples
Chironomidae	64
Entomobryidae	34
Hominidae	7
Sciaridae	7
Agromyzidae	4
Coccinellidae	3
None	2
Psychodidae	2

Outline: negative controls with contaminants
Colour of the oultine indicates partners to track the samples between partner and UMI plates.
Thicker chartreuse outline: FAILED negative controls with contaminants [2%]
Numbers indicate the read count
Squares that are not outlined represent potential sources of contamination within plates: identical sequences found within these wells and negative controls.

Assessment of primary and secondary sequences

NOTE: Controls are not included! 

Number of wells with a primary sequence only: 3430 
Number of wells with primary and secondary sequences: 4793 

Number of primary chimeric sequences: 28 
Number of secondary chimeric sequences: 5496 

NOTE: All secondary chimeric sequences successfully removed

[1] 2
Number of samples with only primary chimeric sequence recognised: 12 
We do not know how mBRAVE recognises chimeras - for now ony samples represented by less than 5 reads get removed
Retained samples: 2

Number of EXCLUDED primary sequences: 469 
 which constitutes 5.71045902836966 % of all samples 
These samples are not being removed - it's an mBRAVE cut-off 

Number of primary sequences with no taxonomy assigned: 110 
 which constitutes 1.33934007061975 % of all samples 
These samples are going to be examined further

Number of samples with no taxonomy assigned that will be replaced with the secondary sequence based on the sequence similarity: 19 
Other sequences with no taxonomy assigned to the primary sequence will remain unchanged.

If the first entry is not ‘Arthropod’, then the second entry is likely correct [based on manual observations]

Number of samples with Wolbachia detected: 421 

Table with plate positions, number of reads, and sequences saved to the output directory:
 /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch19/

Number of samples with Nematoda, Tardigrada, Annelida, and/or Rotifera detected: 71 

Table with plate positions, number of reads, and sequences saved to the output directory:
 /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch19/

Taxon	Frequency
Chordata	38
Nematoda	2
Proteobacteria	64
Rotifera	1

105 wells had primary non-Arthropod hits and secondary Arthropod hits 
NOTE: Primary hits are going to be replaced
 
Samples with only non-Arthropod sequences detected: 56 
NOTE: These samples have been excluded!

Total number of wells with Anopheles reads: 82

Plate	Sequence	No. samples with Anopheles reads
CAMP_014	secondary	1
CAMP_027	primary	8
CAMP_027	secondary	1
CAMP_038	primary	1
CAMP_038	secondary	2
CAMP_039	primary	5
CAMP_039	secondary	6
CAMP_040	primary	4
CAMP_040	secondary	2
CAMP_041	primary	4
CAMP_041	secondary	4
CAMP_042	primary	10
CAMP_043	primary	15
CAMP_043	secondary	3
CAMP_044	primary	4
CAMP_044	secondary	2
CAMP_045	primary	1
CAMP_046	primary	3
CAMP_046	secondary	1
SNST_007	primary	3
SNST_007	secondary	1
WWTS_003	secondary	1

Partner	Plate	No. Anopheles primary sequences with 200 + reads
CAMP	043	15
CAMP	042	10
CAMP	027	8
CAMP	040	4
CAMP	039	3
CAMP	044	3
CAMP	046	3
CAMP	041	2
CAMP	038	1
CAMP	045	1

Number of primary African Anopheline hits [200 or more reads]: 50 
NOTE: All primary mosquito samples removed!

Number of samples with only primary Arthropod sequence: 6290 
 79.309040474089 % of all remaining samples 

Number of samples where secondary sequence is not present elsewhere on the partner or UMI plate: 0 
Number of conflicting sequences [sequences are in different families or orders, both have good read support]: 130

Primary hit	Number
Arthropoda	7830
None	84

Number of retained samples: 7914 
Number of Arthropod samples assigned by mBRAVE [this inscludes samples with fewer than 5 reads that have now been excluded!]: 7983 
Number of samples with replaced sequences: 37 
Retained chimeras: 11 
Retained samples with no taxonomy: 84 

Each retreived sample has only one sequence: TRUE

Number of samples	Description	Category	Decision
5262	Only one sequence with more than 200 reads, no secondary sequence detected	1	YES
814	Only one sequence with 50 to 200 reads, no secondary sequence detected	2	YES
202	Only one sequence with 5 or more but less than 50 reads, no secondary sequence detected	3	YES
120	Dominant sequence with more than 200 reads, non-conflicting secondary sequences with 5 or less reads	4	YES
34	Dominant sequence with 50 to 200 reads, non-conflicting secondary sequences with 5 or less reads	5	YES
759	Dominant sequence with more than 200 reads, conflicting secondary sequences with 5 or less reads	6	YES
316	Dominant sequence with 50 to 200 reads, conflicting secondary sequences with 5 or less read	7	YES
170	Dominant sequence with more than 200 reads, secondary sequences with more than 5 read support	8	NO
140	Dominant sequence with 50 to 200 reads, secondary sequences with more than 5 read support	9	NO
60	Dominant sequence with 5 or more but less than 50 reads, non-conflicting secondary sequences with less than 5 reads	10	NO
37	Dominant sequence with more than 5 but less than 50 reads, any other secondary reads present	11	NO

Decision category	Number of samples
NO	407
YES	7507

8.19025522041764 % OF SAMPLES EXCLUDED [all samples]

12.9118329466357 % OF SAMPLES EXCLUDED [only approved samples]

Plate heatmaps - retained samples

NOTE: The heatmaps below show only the retained samples. Controls, chimeric samples, non-Arthropod samples, and samples with no taxonomy assigned have been removed or replaced!

Final fasta file succesfully saved: /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch19/BOLD_filtered_sequences_batch19.fasta 
Final metadata file succesfully saved: /nfs/users/nfs_a/aw43/aw43/2024_07_bioscan_qc/input/output/qc_reports/batch19/BOLDfiltered_metadata_batch19.csv

The report and output files have been successfully generated!

Number of retained samples per partner plate

Plate	Original number of samples	No. samples post-QC	No. confident samples	Percentage of confident samples
PBRI_010	30	30	30	100.00000
NENM_036	93	92	92	98.92473
NENM_038	93	92	92	98.92473
YARN_026	93	92	92	98.92473
NENM_035	93	91	91	97.84946
YARN_024	93	91	91	97.84946
YARN_028	93	91	91	97.84946
FACE_238	93	90	90	96.77419
PBRI_002	93	91	90	96.77419
PBRI_008	93	92	90	96.77419
SHAP_026	93	92	90	96.77419
YARN_027	93	92	90	96.77419
FACE_239	93	91	89	95.69892
SHAP_025	93	91	89	95.69892
SNST_004	93	93	89	95.69892
YARN_025	93	89	89	95.69892
YARN_029	92	89	88	95.65217
SNST_001	89	87	85	95.50562
SNST_003	94	92	89	94.68085
FACE_229	93	89	88	94.62366
PBRI_007	93	89	88	94.62366
SNST_002	92	89	87	94.56522
SNST_005	92	88	87	94.56522
CAMP_013	94	94	88	93.61702
FACE_240	93	88	87	93.54839
FACE_241	93	90	87	93.54839
NENM_029	93	87	87	93.54839
NENM_031	93	87	87	93.54839
NENM_033	93	87	87	93.54839
NENM_037	93	88	87	93.54839
SNST_006	93	92	87	93.54839
CAMP_048	93	90	86	92.47312
FACE_237	93	90	86	92.47312
NENM_028	93	86	86	92.47312
NENM_030	93	86	86	92.47312
NENM_032	93	86	86	92.47312
WWTS_009	93	89	86	92.47312
CAMP_038	93	88	85	91.39785
FACE_228	93	87	85	91.39785
FACE_235	93	85	85	91.39785
PBRI_006	93	86	85	91.39785
CAMP_047	93	91	84	90.32258
FACE_219	93	88	84	90.32258
NENM_027	93	84	84	90.32258
WWTS_004	93	90	84	90.32258
CAMP_014	94	87	84	89.36170
CAMP_017	94	92	84	89.36170
CAMP_042	93	83	83	89.24731
SNST_007	34	30	30	88.23529
FACE_231	93	88	82	88.17204
FACE_234	93	85	82	88.17204
NENM_039	93	82	82	88.17204
WWTS_005	93	91	82	88.17204
CAMP_026	74	66	65	87.83784
CAMP_045	32	29	28	87.50000
FACE_233	93	86	81	87.09677
FACE_236	93	88	81	87.09677
CAMP_027	74	65	64	86.48649
FACE_222	93	88	80	86.02151
PBRI_001	93	85	80	86.02151
PBRI_004	93	82	80	86.02151
CAMP_015	94	91	80	85.10638
FACE_225	93	83	79	84.94624
NENM_034	93	83	79	84.94624
WWTS_008	93	84	79	84.94624
PBRI_005	93	83	78	83.87097
FACE_220	91	80	76	83.51648
CAMP_018	94	89	78	82.97872
CAMP_019	47	46	39	82.97872
CAMP_044	93	83	77	82.79570
FACE_221	93	83	77	82.79570
YARN_032	93	78	77	82.79570
CAMP_041	93	84	76	81.72043
WWTS_006	93	87	76	81.72043
WWTS_010	83	74	67	80.72289
FACE_209	93	84	75	80.64516
FACE_232	93	82	75	80.64516
PBRI_009	93	91	75	80.64516
WWTS_001	93	90	75	80.64516
WWTS_007	93	83	75	80.64516
YARN_030	93	81	74	79.56989
WWTS_003	93	89	73	78.49462
CAMP_040	93	77	72	77.41935
FACE_223	93	83	72	77.41935
YARN_031	93	74	72	77.41935
CAMP_016	94	87	72	76.59574
CAMP_043	93	74	71	76.34409
CAMP_039	93	81	70	75.26882
FACE_230	93	70	69	74.19355
CAMP_046	93	72	68	73.11828
FACE_224	93	80	68	73.11828
PBRI_003	93	76	68	73.11828
FACE_226	93	66	62	66.66667
WWTS_002	93	82	58	62.36559
FACE_227	93	55	51	54.83871
NENM_040	64	20	20	31.25000

Number of retained samples per partner

Partner	Original number of samples	No. samples post-QC	No. confident samples	Percentage of confident samples
SHAP	186	183	179	96.23656
SNST	587	571	554	94.37819
YARN	836	777	764	91.38756
NENM	1273	1151	1146	90.02357
PBRI	867	805	764	88.11995
FACE	2230	1999	1891	84.79821
CAMP	1721	1569	1454	84.48576
WWTS	920	859	755	82.06522

Number of retained samples per UMI plate

Plate	Original number of samples	No. samples post-QC	No. confident samples	Percentage of confident samples
22	371	364	361	97.30458
10	372	354	353	94.89247
8	372	359	353	94.89247
16	312	303	293	93.91026
24	372	364	349	93.81720
19	372	343	343	92.20430
21	362	343	333	91.98895
9	311	290	285	91.63987
15	368	355	337	91.57609
7	372	348	334	89.78495
12	372	340	331	88.97849
20	372	340	327	87.90323
13	309	303	270	87.37864
17	376	359	324	86.17021
18	309	292	265	85.76052
3	370	335	312	84.32432
2	353	306	296	83.85269
6	372	326	307	82.52688
1	372	330	303	81.45161
4	372	334	299	80.37634
14	372	352	297	79.83871
23	372	305	291	78.22581
5	372	297	286	76.88172
11	343	272	258	75.21866

The plates with low number of reads and retained samples should be examined!

Samples to examine manually

Failed negative controls [2%] with contamination other than Bovidae:

 CONTROL_NEG_LYSATE_CAMP_016_H12
CONTROL_NEG_LYSATE_CAMP_017_H12
CONTROL_NEG_LYSATE_FACE_222_H12

These samples may have insects in them!